Pocket Modification of x-Amine Transaminase AtATA for Overcoming the Trade-Off Between Activity and Stability Toward 1-Acetonaphthone

Amine transaminases(ATAs)catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines.However,the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of ATAs.Overcoming this trade-off is important for developing robustly engineered enzymes and a universal approach for ATAs.Herein,we modified the binding pocket of co-ATA from Aspergillus terreus(AtATA)to identify the key amino acid residues controlling the activity and stability of AtATA toward 1-acetonaphthone.We discovered a structural switch comprising four key amino acid sites(R128,V149,L182,and L187),as well as the"best"mutant(AtATAD224K/V149A/L182 F/L187F;termed M4).Compared to the parent enzyme AtATAD224K(AtATAPa),M4 increased the catalytic efficiency(k_(cat)/K_(m)^(1-acetonaphthone),where kcatis the constant of catalytic activities and is 10.1 min^(-1),K_(m)^(1-acetonaphthoneis) Michaelis-Menten constant and is 1.7 mmol·L^(-1))and half-life(t1/2)by 59-fold to 5.9 L·min^(-1)·mmol-1and by 1.6-fold to 46.9 min,respectively.Moreover,using M4 as the biocatalyst,we converted a 20 mmol·L^(-1)aliquot of 1-acetonaphthone in a 50 mL scaled-up system to the desired product,(R)-(+)-1(1-naphthyl)ethylamine((R)-NEA),with 78%yield and high enantiomeric purity(R>99.5%)within 10 h.M4 also displayed significantly enhanced activity toward various 1-acetonaphthone analogs.The related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and thermostability.Strengthening of intramolecular interactions and expansion of the angle between the substratebinding pocket and the pyridoxal 5’-phosphate(PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and activity.Moreover,this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs,which exhibited 8%-22%sequence similarity with AtATA.This research has important implications for overcoming the trade-off between ATA activity and thermostability.

Electron engineering of nickel phosphide for Niδ+in electrochemical nitrate reduction to ammonia

The electrochemical reduction of nitrate to ammonia(ENRA)provides an efficient approach to remove nitrate pollution and achieve ammonia production simultaneously.Herein,inspired by bio-enzyme in denitrifying bacteria,a carbon-coated nickel phosphide(NiPC)nanosheet derived from metal-organic frameworks(MOFs)is proposed as an efficient catalyst for ENRA.Through electron engineering,controllable Niδ+in nickel phosphide is achieved by regulating the degree of phosphating,which enhances its activity for the hydrogenation of nitrate.As the result,Niδ+becomes one of dominating factors determining the efficiency of the ENRA reaction in nickel phosphide.The optimal NiPC catalyst exhibits impressive property toward ENRA:NH_(4)^(+)Faraday efficiency of 96.68%,NH4+selectivity of 99.04%,and nitrate conversion rate of 90.43%under low nitrate concentration(200 mg·L^(−1)).This work opens a new avenue for the design of next-generation catalysts through electron engineering for ENRA.

Mangiferin alleviates renal infammatory injury in spontaneously hypertensive rats by inhibiting MCP-1/CCR2 signaling pathway

Objective: Hypertension is a low-grade infammation state of the disease and was easily complicated by kidneys’ infammatory response. Mangiferin(MGF), a pharmacologically active compound in various plants including Mangifera indica, has a strong anti-infammatory activity. However, the effects of MGF on renal infammatory injury in spontaneously hypertensive rats(SHRs) remain unclear. The purpose of this study was to investigate the protective effects and mechanisms of MGF on renal infammatory injury in SHRs.Methods: MGF was used in SHRs at the doses of 10, 20, 40 mg/kg/d for 8 weeks consecutively. The blood and urine were collected for assessment of renal function. Renal tissues were collected for histological,immunohistochemistry, ELISA, Western blot and real time reverse transcription PCR(RT-PCR) analysis.Results: The results showed that the levels of interleukin 6(IL-6), tumor necrosis factor-a(TNF-a), monocyte chemoattractant protein-1(MCP-1) and recombinant chemokine C-C-Motif receptor 2(CCR2) were increased in SHRs, meanwhile, the level of IL-10 was decreased in SHR. Treatment of MGF inhibited the expression of IL-6, TNF-a, MCP-1 and CCR2, and promoted the expression of IL-10. Furthermore, the content of blood urea nitrogen(BUN) and serum uric acid(SUA) was significantly increased in the model group, and treatment of MGF had no obvious effects on these parameters at all dose levels.Conclusion: Our study proved that the kidneys of SHRs had significant infammatory injury, and MGF had the protective effects on renal infammatory injury in SHRs;The protective mechanism may be mediated partly by the MCP-1/CCR2 signaling pathway. Thus, it is a potential new drug for the treatment of hypertension.